Role of presynaptic targeting of alpha-synuclein in the pathogenesis of Parkinson's Disease and Dementia with Lewy Bodies

α-突触核蛋白突触前靶向在帕金森病和路易体痴呆发病机制中的作用

• 批准号: 9886289
• 负责人: Laura A. Volpicelli-Daley
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9886289
• 关键词: Adult; Amygdaloid structure; Antisense Oligonucleotides; Biochemistry; Brain; Brain region; Cell membrane; Cells; Confocal Microscopy; Corpus striatum structure; Cytoplasmic Inclusion; Data; Development; Disease; Disease Progression; Dominant-Negative Mutation; Family; Fluorescence Recovery After Photobleaching; Genes; Goals; Idiopathic Parkinson Disease; Immunohistochemistry; Immunoprecipitation; Injections; Knock-in Mouse; LRRK2 gene; Leucine-Rich Repeat; Lewy Bodies; Lewy Body Dementia; Lewy neurites; MYO5A gene; Membrane; Methods; Modeling; Motor; Mus; Mutation; Myosin ATPase; Myosin Type V; Neurons; Organelles; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathologic; Pathway interactions; Patients; Peptides; Phosphotransferases; Physiological; Play; Presynaptic Terminals; Protein Isoforms; Proteins; Research; Risk Factors; Role; Specific qualifier value; Specificity; Subfamily lentivirinae; Substantia nigra structure; Synapses; Synapsins; Synaptic Vesicles; Testing; Toxic effect; Ventricular; Vesicle; adeno-associated viral vector; alpha synuclein; cell type; dopaminergic neuron; in vivo; inhibitor/antagonist; kinase inhibitor; member; mutant; new therapeutic target; novel; overexpression; pars compacta; preclinical development; presynaptic; prevent; rab GTP-Binding Proteins; small hairpin RNA; therapeutic target; trafficking; vector

Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB) are characterized by cytoplasmic inclusions called Lewy Bodies and Lewy Neurites which are composed primarily of α-synuclein (α-syn). Multiple lines of evidence suggest that these inclusions contribute to disease pathogenesis. Thus, preventing α-syn aggregation is a critical therapeutic target for preventing disease progression. Normally, α-syn resides at the presynaptic terminal, preferentially associates with synaptic vesicles, and interaction of α-syn with membranes inhibit its aggregation. Our proposed study seeks to determine how presynaptic targeting of α-syn prevents it from converting to a pathologic, aggregation prone form and how PD risk factors disrupt the normal localization of α-syn and increase its propensity to forming inclusions. Mutations in leucine rich repeat kinase (LRRK2) are the most common cause of familial Parkinson’s disease. We showed that the G2019S-LRRK2 mutations increase the formation of α-syn inclusions and that LRRK2 kinase inhibitors reduce inclusion formation, but the mechanisms by which LRRK2 controls the propensity of α-syn to form aggregates is unknown. Although current research focuses on the role of LRRK2 in degradative organelles, LRRK2 has also been functionally implicated at the presynaptic terminal and may play a role in α-syn presynaptic membrane targeting. Recently, a subset of Rab GTPases was identified as LRRK2 substrates. Rabs control distinct steps in membrane trafficking pathways in the cell. Ten years ago, the Lindquist lab identified the same Rabs (that were recently shown to be phosphorylated by LRRK2) as protective in models of α-syn toxicity. We will determine if Rabs phosphorylated by LRRK2 enhance presynaptic targeting of α-syn and prevent α-syn inclusion formation. Because inclusions localize to multiple brain regions in PD and DLB, we will inject fibrils into the mouse striatum which produces inclusions in the substantia nigra pars compacta (SNpc), cortex, and amygdala. In Aim 1, we will determine the extent to which select Rab isoforms influence α-syn presynaptic targeting using novel AAV vectors to increase Rab expression in the brain or use antisense oligonucleotides (ASOs) to reduce Rab levels. Immunohistochemistry and biochemistry will be used to determine if expression of these Rabs in vivo prevents formation of fibril-induced inclusions in multiple brain regions, and loss of dopamine neurons in the SNpc. In Aim 2, we will identify the Rab effectors in neurons that specify targeting of vesicles containing α- syn cargo to the presynaptic terminal. Finally, in Aim 3, we will determine if LRRK2 kinase activity prevents presynaptic targeting of α-syn and if the mislocalization of α-syn in the cell increases its propensity to form inclusions. We will also determine the extent to which LRRK2 kinase inhibitors and ASOs in preclinical development enhance presynaptic targeting of α-syn. These studies will open up new research avenues for understanding how factors and genes that cause PD and DLB increase the propensity of α-syn to aggregate, and how to therapeutically target LRRK2 and downstream effectors to prevent the progression of PD.

帕金森病（PD）和路易体痴呆（DLB）的特征是细胞质内含物 称为路易体和路易神经突，主要由α-突触核蛋白（α-syn）组成。多行 有证据表明这些内含物有助于疾病的发病机理。因此，防止α-syn 聚集是预防疾病进展的关键治疗靶点。通常，α-syn位于 突触前末梢，优先与突触囊泡结合，以及α-syn与膜的相互作用 抑制其聚集。我们提出的研究旨在确定α-syn的突触前靶向如何阻止它 从转化为病理性聚集倾向形式，以及PD风险因素如何破坏正常定位 α-syn和增加其倾向形成夹杂物。富含亮氨酸重复序列激酶（LRRK 2）的突变是 家族性帕金森病最常见的病因我们发现G2019 S-LRRK 2突变 增加α-syn包涵体的形成，LRRK 2激酶抑制剂减少包涵体的形成，但 LRRK 2控制α-syn形成聚集体的倾向的机制尚不清楚。虽然 目前的研究集中在LRRK 2在降解细胞器中的作用，LRRK 2也被功能性地 参与突触前末端，并可能在α-syn突触前膜靶向中发挥作用。最近， Rab GTP酶的一个亚类被鉴定为LRRK 2底物。Rabs控制膜中的不同步骤 细胞中的运输途径。十年前，林德奎斯特实验室发现了同样的Rabs（最近被发现）。 显示被LRRK 2磷酸化）在α-syn毒性模型中具有保护作用。我们将确定， 通过LRRK 2磷酸化增强α-syn的突触前靶向并防止α-syn包含物形成。 由于包涵体定位于PD和DLB的多个脑区，我们将原纤维注射到小鼠体内， 纹状体，其在黑质部（SNpc）、皮质和杏仁核中产生内含物。在 目的1，我们将确定选择Rab亚型影响α-syn突触前靶向的程度， 新的AAV载体来增加Rab在脑中的表达或使用反义寡核苷酸（ASO）来减少Rab在脑中的表达。 Rab水平。将使用免疫组织化学和生物化学来确定这些Rab的表达是否在 体内防止在多个脑区域形成纤维诱导的包涵体，以及在脑内多巴胺神经元的损失。 SNPC。在目标2中，我们将鉴定神经元中的Rab效应子，其指定含有α- 突触前末梢的突触货物。最后，在目标3中，我们将确定LRRK 2激酶活性是否阻止 如果α-syn在细胞中的错误定位增加了其形成突触前靶向的倾向， 内含物。我们还将确定LRRK 2激酶抑制剂和ASO在临床前治疗中的作用程度。 发育增强α-syn的突触前靶向。这些研究将开辟新的研究途径， 了解导致PD和DLB的因素和基因如何增加α-syn聚集的倾向， 以及如何治疗性靶向LRRK 2和下游效应物以防止PD的进展。